E3/E4 Mice Meningeal Dura scRNAseq

QC and DE

Setup

Libraries

# load packages
library(ComplexUpset) 
library(dplyr)        # ungroup()
library(ggrepel)      # geom_text_repel()
library(ggtree)       # BuildClusterTree()
library(gridExtra)    # grid.arrange()
library(gtools)       # smartbind()
library(parallel)     # detectCores()
library(plotly)       # plot_ly()
library(Seurat)       # DimPlot()\
library(tidyr)        # %>%
library(UpSetR)       # fromList()

Variables and functions

out <- "../../results/pass_1/all_clusters/"
sample_order <- c("E3.M","E3.F","E4.M","E4.F")
sample_colors <- c("#26946A","#1814A1","#EAC941","#DF5F00")
sample_order2 <- c("Male E3", "Male E4", "Female E3", "Female E4")
isoform_order <- c("E4","E3")
isoform_colors <- c("darkgray","cornflowerblue")
sex_order <- c("Male","Female")
sex_colors <- c("darkgray","purple")

Load data

mouse.annotated <- readRDS("../../rObjects/annotated_obj.rds")

QC

2D UMAP

# manually set colors
cluster_colors <- c("#B5B9BA", # Pericytes and SMCs
                    "#40BBFF", # Endothelial
                    "#a5d5a9", # B cells
                    "#5dbd64", # Plasma cells
                    "#1c7e24", # T and NK cells
                    "#F57C7C", # ILCs
                    "#E42622", # Dendritic cells
                    "#FBB268", # Neutrophils
                    "#FE8D19", # Macrophages
                    "#A6CEE3", # Mast cells
                    "#9D7BBA", # Fibroblasts
                    "#977899") # Schwann cells

# plot umap
umap <- DimPlot(object = mouse.annotated, 
                reduction = "umap",
                repel = TRUE,
                group.by = "annotated_clusters",
                cols = cluster_colors)
umap

3D UMAP

embeddings <- mouse.annotated@reductions$umap@cell.embeddings
embeddings <- cbind(embeddings, as.character(mouse.annotated$annotated_clusters))
colnames(embeddings)[4] <- "annotated_clusters"
embeddings <- as.data.frame(embeddings)

three.dim <- plot_ly(embeddings,
                     x = ~umap_1, 
                     y = ~umap_2, 
                     z = ~umap_3, 
                     color = ~annotated_clusters, 
                     colors = cluster_colors,
                     size = 1) 
three.dim <- three.dim %>% add_markers() 
three.dim <- three.dim %>% layout(scene = list(xaxis = list(title = 'UMAP_1'), 
                                     yaxis = list(title = 'UMAP_2'), 
                                     zaxis = list(title = 'UMAP_3')))
three.dim

Cluster markers

goi <- c("Pdgfrb","Acta2","Vwf","Cldn5","Pecam1","Flt4","Prox1","Lyve1","Ptprc",
         "Cd19","Ms4a1","Ighd","Cd3e","Trbc2","Il7r","Nkg7","Klrb1b","Klrb1c",
         "Gata3","Rora","Itgax","H2-Eb1","Ccr2","Ly6c2","Lyz2","Ly6g","Itgam",
         "Mrc1","Csf1r","Cd38","Mki67","Mcpt4","Ms4a2","Col1a2","Plp1")

v <- VlnPlot(mouse.annotated,
             features = goi,
             split.by = "annotated_clusters",
             flip = TRUE,
             stack = TRUE,
             cols = cluster_colors)

## The default behaviour of split.by has changed.
## Separate violin plots are now plotted side-by-side.
## To restore the old behaviour of a single split violin,
## set split.plot = TRUE.
##       
## This message will be shown once per session.

v

Split UMAP

# Apoe isoform
umap1 <- DimPlot(object = mouse.annotated, 
                 group.by = "annotated_clusters",
                 reduction = "umap",
                 split.by = "isoform",
                 repel = TRUE,
                 cols = cluster_colors)
umap1

# sex
umap2 <- DimPlot(object = mouse.annotated, 
                 group.by = "annotated_clusters",
                 reduction = "umap",
                 split.by = "sex",
                 repel = TRUE,
                 cols = cluster_colors)
umap2

# sample
umap3 <- DimPlot(object = mouse.annotated, 
                 group.by = "annotated_clusters",
                 reduction = "umap",
                 split.by = "sample",
                 ncol = 2,
                 repel = TRUE,
                 cols = cluster_colors)
umap3

# phase
umap4 <- DimPlot(object = mouse.annotated, 
                 group.by = "annotated_clusters",
                 reduction = "umap",
                 split.by = "phase",
                 cols = cluster_colors,
                 repel = TRUE)
umap4

# mito.factor
umap5 <- DimPlot(object = mouse.annotated, 
                 group.by = "annotated_clusters",
                 reduction = "umap",
                 split.by = "mito.factor",
                 cols = cluster_colors,
                 ncol = 2,
                 repel = TRUE)
umap5

# sample2
umap6 <- DimPlot(object = mouse.annotated, 
                 group.by = "annotated_clusters",
                 reduction = "umap",
                 split.by = "sample2",
                 cols = cluster_colors,
                 ncol = 2,
                 repel = TRUE)
umap6

Heatmap UMAP

# UMAP percent.mt
f1 <- FeaturePlot(mouse.annotated, 
            reduction = "umap", 
            features = "percent.mt")  + 
  scale_colour_gradientn(colours = c("blue","lightblue","yellow","orange","red"))
f1

# UMAP nCount
f2 <- FeaturePlot(mouse.annotated, 
            reduction = "umap",
            features = "nCount_RNA") + 
  scale_colour_gradientn(colours = c("blue","lightblue","yellow","orange","red"))
f2

# UMAP nFeature
f3 <- FeaturePlot(mouse.annotated, 
            reduction = "umap", 
            features = "nFeature_RNA") + 
  scale_colour_gradientn(colours = c("blue","lightblue","yellow","orange","red"))
f3

# UMAP percent.ribo
f4 <- FeaturePlot(mouse.annotated, 
            reduction = "umap", 
            features = "percent.ribo.protein") + 
  scale_colour_gradientn(colours = c("blue","lightblue","yellow","orange","red"))
f4

# UMAP cell.complexity
f5 <- FeaturePlot(mouse.annotated, 
            reduction = "umap", 
            features = "cell.complexity") + 
  scale_colour_gradientn(colours = c("blue","lightblue","yellow","orange","red"))
f5

Percent cells per cluster

# isoform
b1 <- mouse.annotated@meta.data %>%
  group_by(annotated_clusters, isoform) %>%
  dplyr::count() %>%
  group_by(annotated_clusters) %>%
  dplyr::mutate(percent = 100*n/sum(n)) %>%
  ungroup() %>%
  ggplot(aes(x=annotated_clusters,y=percent, fill=isoform)) +
  theme_classic() +
  geom_col() +
  scale_fill_manual(values = isoform_colors) +
  ggtitle("Percentage of isoform per cluster") +
  theme(axis.text.x = element_text(angle = 90,vjust = 0.5, hjust=1))
b1

# sex
b2 <- mouse.annotated@meta.data %>%
  group_by(annotated_clusters, sex) %>%
  dplyr::count() %>%
  group_by(annotated_clusters) %>%
  dplyr::mutate(percent = 100*n/sum(n)) %>%
  ungroup() %>%
  ggplot(aes(x=annotated_clusters,y=percent, fill=sex)) +
  theme_classic() +
  geom_col() +
  scale_fill_manual(values = sex_colors) +
  ggtitle("Percentage of sex per cluster") +
  theme(axis.text.x = element_text(angle = 90,vjust = 0.5, hjust=1))
b2

# sample
b3 <- mouse.annotated@meta.data %>%
  group_by(annotated_clusters, sample) %>%
  dplyr::count() %>%
  group_by(annotated_clusters) %>%
  dplyr::mutate(percent = 100*n/sum(n)) %>%
  ungroup() %>%
  ggplot(aes(x=annotated_clusters,y=percent, fill=sample)) +
  theme_classic() +
  geom_col() +
  scale_fill_manual(values = sample_colors) +
  ggtitle("Percentage of sample per cluster") +
  theme(axis.text.x = element_text(angle = 90,vjust = 0.5, hjust=1))
b3

# phase
b4 <- mouse.annotated@meta.data %>%
  group_by(annotated_clusters, phase) %>%
  dplyr::count() %>%
  group_by(annotated_clusters) %>%
  dplyr::mutate(percent = 100*n/sum(n)) %>%
  ungroup() %>%
  ggplot(aes(x=annotated_clusters,y=percent, fill=phase)) +
  theme_classic() +
  geom_col() +
  ggtitle("Percentage of phase per cluster") +
  theme(axis.text.x = element_text(angle = 90,vjust = 0.5, hjust=1))
b4

# mito.factor
b5 <- mouse.annotated@meta.data %>%
  group_by(annotated_clusters, mito.factor) %>%
  dplyr::count() %>%
  group_by(annotated_clusters) %>%
  dplyr::mutate(percent = 100*n/sum(n)) %>%
  ungroup() %>%
  ggplot(aes(x=annotated_clusters,y=percent, fill=mito.factor)) +
  theme_classic() +
  geom_col() +
  ggtitle("Percentage of mito.factor per cluster") +
  theme(axis.text.x = element_text(angle = 90,vjust = 0.5, hjust=1))
b5

Cluster tree

# build tree
Idents(mouse.annotated) <- mouse.annotated$annotated_clusters
mouse.annotated <- BuildClusterTree(object = mouse.annotated,
                                     dims = 1:15,
                                     reorder = FALSE,
                                     reorder.numeric = FALSE)

tree <- mouse.annotated@tools$BuildClusterTree
tree$tip.label <- paste0(tree$tip.label)

# plot tree
p <- ggtree::ggtree(tree, aes(x, y)) +
  scale_y_reverse() +
  ggtree::geom_tree() +
  ggtree::theme_tree() +
  ggtree::geom_tiplab(offset = 1) +
  ggtree::geom_tippoint(color = cluster_colors[1:length(tree$tip.label)], shape = 16, size = 5) +
  coord_cartesian(clip = 'off') +
  theme(plot.margin = unit(c(0,2.5,0,0), 'cm'))
p

Cells per sample

# extract data
data <- as.data.frame(table(mouse.annotated$sample))
colnames(data) <- c("sample","frequency")

# plot
ncells <- ggplot(data, aes(x = sample, y = frequency, fill = sample)) + 
  geom_col() +
  theme_classic() +
  geom_text(aes(label = frequency), 
            position=position_dodge(width=0.9), 
            vjust=-0.25) +
  scale_fill_manual(values = sample_colors) + 
  scale_y_continuous(breaks = seq(0,8000, by = 1000), limits = c(0,8000)) +
  ggtitle("Filtered: cells per sample") +
  theme(legend.position =  "none") + 
  theme(axis.text.x = element_text(angle = 45, hjust=1))
ncells

Cells per cluster

# extract data
data <- as.data.frame(table(mouse.annotated$annotated_clusters))
colnames(data) <- c("cluster","frequency")

# plot
ncells <- ggplot(data, aes(x = cluster, y = frequency, fill = cluster)) + 
  geom_col() +
  theme_classic() +
  geom_text(aes(label = frequency), 
            position=position_dodge(width=0.9), 
            vjust=-0.25) +
  scale_fill_manual(values = cluster_colors) + 
  scale_y_continuous(breaks = seq(0,5000, by = 1000), limits = c(0,5000)) +
  ggtitle("Cells per Cluster") +
  theme(legend.position =  "none") + 
  theme(axis.text.x = element_text(angle = 45, hjust=1))
ncells

Differential expression

E4 female vs E3 female within each cluster

# initialize variables
cell_types <- levels(mouse.annotated$annotated_clusters)
female.df <- data.frame()

# loop through clusters
for (i in cell_types) {
  print(i)
  cluster <- subset(mouse.annotated, annotated_clusters == i)
  Idents(cluster) <- cluster$sample
  markers <- FindMarkers(object = cluster,
                         ident.1 = "E4.F",
                         ident.2 = "E3.F",
                         only.pos = FALSE, # default
                         min.pct = 0.10,  # default
                         test.use = "MAST",
                         verbose = TRUE,
                         assay = "RNA")
  if(nrow(markers) == 0) {
    next
  }
  markers$cluster <- i
  markers$gene <- rownames(markers)
  female.df <- rbind(female.df, markers)
}

# reformat table
colnames(female.df)[c(3,4)] <- c("percent_E4_F","percent_E3_F")
rownames(female.df) <- 1:nrow(female.df)
female.df$percent_difference <- abs(female.df$percent_E4_F - female.df$percent_E3_F)
female.df <- female.df[,c(6,7,1,5,2,3,4,8)]

# write table
write.table(female.df, 
            file = paste0(out, "DEGs/DEG_tables/E4_F_vs_E3_F_DEGs.tsv"), 
            sep = "\t", quote = FALSE, row.names = FALSE)

E4 male vs E3 male within each cluster

# initialize variable
cell_types <- levels(mouse.annotated$annotated_clusters)
male.df <- data.frame()

# loop through clusters
for (i in cell_types) {
  print(i)
  cluster <- subset(mouse.annotated, annotated_clusters == i)
  Idents(cluster) <- cluster$sample
  markers <- FindMarkers(object = cluster,
                         ident.1 = "E4.M",
                         ident.2 = "E3.M",
                         only.pos = FALSE, # default
                         min.pct = 0.10,  # default
                         test.use = "MAST",
                         verbose = TRUE,
                         assay = "RNA")
  if(nrow(markers) == 0) {
    next
  }
  markers$cluster <- i
  markers$gene <- rownames(markers)
  male.df <- rbind(male.df, markers)
}

# reformat table
colnames(male.df)[c(3,4)] <- c("percent_E4_M","percent_E3_M")
rownames(male.df) <- 1:nrow(male.df)
male.df$percent_difference <- abs(male.df$percent_E4_M - male.df$percent_E3_M)
male.df <- male.df[,c(6,7,1,5,2,3,4,8)]

# write table
write.table(male.df, 
            file = paste0(out, "DEGs/DEG_tables/E4_M_vs_E3_M_DEGs.tsv"), 
            sep = "\t",quote = FALSE, row.names = FALSE)

Compare DEGs

# read tables
female.df <- read.table(paste0(out, "DEGs/DEG_tables/E4_F_vs_E3_F_DEGs.tsv"),
                        sep = "\t", 
                        header = TRUE)
male.df <- read.table(paste0(out, "DEGs/DEG_tables/E4_M_vs_E3_M_DEGs.tsv"),
                      sep = "\t", 
                      header = TRUE)

# filter
female.df <- female.df[female.df$p_val_adj < 0.05,]
male.df <- male.df[male.df$p_val_adj < 0.05,]

# add columns
direction <- female.df$avg_log2FC > 0
direction <- gsub(TRUE, "E4_female_up", direction)
direction <- gsub(FALSE, "E4_female_down", direction)
female.df$direction <- direction
direction <- male.df$avg_log2FC > 0
direction <- gsub(TRUE, "E4_male_up", direction)
direction <- gsub(FALSE, "E4_male_down", direction)
male.df$direction <- direction

# reformat tables
female.df2 <- female.df %>%
  dplyr::count(cluster,direction) %>%
  tidyr::spread(cluster, n)
male.df2 <- male.df %>%
  dplyr::count(cluster,direction) %>%
  tidyr::spread(cluster, n)

# master table
df <- smartbind(female.df2, male.df2)

# save
write.table(df, 
            file = paste0(out, "DEGs/DEG_tables/DEG_comparison_pvaladj_0.05.tsv"),
            sep = "\t", 
            quote = FALSE, 
            row.names = FALSE)

DEG heatmap

# set heatmap colors and names
paletteLength <- 100
myColor <- colorRampPalette(c("white","#f0eb9e","darkgreen"))(paletteLength)

# reformat table
df2 <- df
rownames(df2) <- df2$direction
df2$direction <- NULL

# save
path <- paste0(out, "DEGs/DEG_tables/DEG_comparison_heatmap_pvaladj_0.05.pdf")
pdf(path, width = 10, height = 6)

# plot
pheatmap::pheatmap(df2,
                   main = "FDRq < 0.05, |LFC| > 0",
                   treeheight_row = 0,
                   treeheight_col = 0,
                   color = myColor,
                   cluster_rows = FALSE,
                   display_numbers = round(df2, digits = 0),
                   fontsize_number = 12,
                   number_color = "black")

Volcano

variables <- c("E4_F_vs_E3_F","E4_M_vs_E3_M")
all_clusters <- levels(mouse.annotated$annotated_clusters)

for (i in variables) {
  
  # read DEG file
  if (i == "E4_F_vs_E3_F") {
    treatment_vs_control <- 
      read.delim(paste0(out, "DEGs/DEG_tables/E4_F_vs_E3_F_DEGs.tsv"), sep = "\t")
  } else {
    treatment_vs_control <-
      read.delim(paste0(out, "DEGs/DEG_tables/E4_M_vs_E3_M_DEGs.tsv"), sep = "\t")
  }
  
  # assign colors
  color_values <- vector()
  max <- nrow(treatment_vs_control)
  for(row in 1:max){
    if (treatment_vs_control$p_val_adj[row] < 0.05){
      if (treatment_vs_control$avg_log2FC [row] > 0){
        color_values <- c(color_values, 1) # 1 when logFC > 0 and FDRq < 0.05
      }
      else if (treatment_vs_control$avg_log2FC[row] < 0){
        color_values <- c(color_values, 2) # 2 when logFC < 0 and FDRq < 0.05
      }
    }
    else{
      color_values <- c(color_values, 3) # 3 when FDRq >= 0.05
    }
  }
  treatment_vs_control$color_adjpval_0.05 <- factor(color_values)
  
  # loop through clusters
  for (j in all_clusters) {
    
    # subset cluster
    data <- subset(treatment_vs_control, cluster == j)
    
    # plot only if there are DEGs with p_val_adj < 0.05
    num <- subset(data, p_val_adj < 0.05)
    num <- nrow(num)
    if(num != 0) {
        
      # subset genes to label
      up <- data[data$color_adjpval_0.05 == 1,]
      up10 <- up[1:10,]
      down <- data[data$color_adjpval_0.05 == 2,]
      down10 <- down[1:10,]
      
      # set manual colors
      if (!1 %in% unique(data$color_adjpval_0.05)) {
        my_colors <- c("blue","gray")
      } else if (!2 %in% unique(data$color_adjpval_0.05)) {
        my_colors <- c("red","gray")
      } else if (!1 %in% unique(data$color_adjpval_0.05) && !2 %in% unique(data$color_adjpval_0.05)) {
        my_colors <- c("gray")
      } else {
        my_colors <- c("red","blue","gray")
      }
      
      # set significance threshold
      hadjpval <- (-log10(max(
        data$p_val[data$p_val_adj < 0.05], 
        na.rm=TRUE)))

      # plot
      p <-
        ggplot(data = data, 
               aes(x = avg_log2FC,  # x-axis is logFC
                   y = -log10(p_val),  # y-axis will be -log10 of P.Value
                   color = color_adjpval_0.05)) +  # color is based on factored color column
        geom_point(alpha = 0.8, size = 2) +  # create scatterplot, alpha makes points transparent
        theme_bw() +  # set color theme
        theme(legend.position = "none") +  # no legend
        scale_color_manual(values = my_colors) +  # set factor colors
        labs(
          title = "", # no main title
          x = expression(log[2](FC)), # x-axis title
          y = expression(-log[10] ~ "(" ~ italic("p") ~ "-value)") # y-axis title
        ) +
        theme(axis.title.x = element_text(size = 10),
              axis.text.x = element_text(size = 10)) +
        theme(axis.title.y = element_text(size = 10),
              axis.text.y = element_text(size = 10)) +
        geom_hline(yintercept = hadjpval,  #  horizontal line
                           colour = "#000000",
                           linetype = "dashed") +
        ggtitle(paste0(j,"\n",i,", p_val_adj < 0.05")) +
        geom_text_repel(data = up10,
                        aes(x = avg_log2FC, y= -log10(p_val), label = gene), 
                        color = "maroon", 
                        fontface="italic",
                        max.overlaps = getOption("ggrepel.max.overlaps", default = 30)
                        ) +
        geom_text_repel(data = down10,
                        aes(x = avg_log2FC, y= -log10(p_val), label = gene), 
                        color = "navyblue", 
                        fontface="italic",
                        max.overlaps = getOption("ggrepel.max.overlaps", default = 30)
                        )
      p
      
      # save
      clus <- j
      clus <- gsub(" ","_",clus)
      clus <- gsub("/","_",clus)
      clus <- gsub("-","_",clus)
      if (i == "E4_F_vs_E3_F") {
        path <- paste0(out, "DEGs/volcano/E4_F_vs_E3_F/", tolower(clus),"_female_volcano")
      } else {
        path <- paste0(out, "DEGs/volcano/E4_M_vs_E3_M/", tolower(clus),"_male_volcano")      
      }
      pdf(paste(path,".pdf"), height = 8, width = 8)
      print(p)
      dev.off()
      
      print(paste("i =",i,", j =",j))
      
    }
  } # end loop through clusters
} # end loop through variables

Metascape input

# set variables
thresh <- 0.05

# get file list
files <- list.files(paste0(out, "DEGs/DEG_tables/"))
keep <- grep("_DEGs.tsv", files)
files <- files[keep]
files <- paste0(out, "DEGs/DEG_tables/", files)

# get cell types
cell_types <- levels(mouse.annotated$annotated_clusters)

# loop through files
for (i in 1:length(files)) {
  
  # read table
  data <- read.table(files[i], header = TRUE, sep = "\t")
  data <- data[data$p_val_adj < thresh,]
  up.df <- data[data$avg_log2FC > 0,]
  down.df <- data[data$avg_log2FC < 0,]
  
  # loop through cluster
  for (j in cell_types) {
    
    # subset based on cluster
    up <- up.df[up.df$cluster == j,]
    up <- up$gene
    down <- down.df[down.df$cluster == j,]
    down <- down$gene
    
    # file name
    clus <- j
    clus <- gsub(" ","_",clus)
    file <- gsub(paste0(out,"DEGs/DEG_tables/"), "", files[i])
    file <- gsub("_DEGs.tsv", "", file)
    file <- paste0(out, "DEGs/metascape_input/", file, "/", clus)
     
    # save
    if(length(up) > 10) {
      write.table(x = up,
                  file = paste0(file, "_up_FDRq_", format(thresh, nsmall = 2), ".tsv"),
                  quote = FALSE,
                  row.names = FALSE,
                  col.names = FALSE)      
    }
    if (length(down) > 10) {
       write.table(x = down,
                file = paste0(file, "_down_FDRq_", format(thresh, nsmall = 2), ".tsv"),
                quote = FALSE,
                row.names = FALSE,
                col.names = FALSE)   
    }
  } # end j for loop
} # end i for loop

Upset plot

# read tables
female.df <- read.table(paste0(out, "DEGs/DEG_tables/E4_F_vs_E3_F_DEGs.tsv"),
                         sep = "\t", 
                        header = TRUE)
male.df <- read.table(paste0(out, "DEGs/DEG_tables/E4_M_vs_E3_M_DEGs.tsv"),
                         sep = "\t", header = TRUE)

# filter tables
female.df <- female.df[female.df$p_val_adj < 0.05,]
male.df <- male.df[male.df$p_val_adj < 0.05,]

# loop through clusters
clusters <- levels(mouse.annotated$annotated_clusters)
for (i in clusters) {
  # Subset df by cluster
  print(i)
  female <- subset(female.df, female.df$cluster == i)
  sex <- subset(male.df, male.df$cluster == i)
  
  # Subset lists
  female_up <- subset(female$gene, female$avg_log2FC > 0)
  female_down <- subset(female$gene, female$avg_log2FC < 0)
  male_up <- subset(sex$gene, sex$avg_log2FC > 0)
  male_down <- subset(sex$gene, sex$avg_log2FC < 0)
  list_input <- list("E4 Female Up-regulated" = female_up,
                     "E4 Male Up-regulated" = male_up,
                     "E4 Female Down-regulated" = female_down,
                     "E4 Male Down-regulated" = male_down)
  data <- fromList(list_input)
  
  # store names
  names <- c("E4 Male Down-regulated","E4 Female Down-regulated",
             "E4 Male Up-regulated","E4 Female Up-regulated")
  
  # plot
  upset_gene <- ComplexUpset::upset(data, 
                      names,
                      set_sizes=(
                        upset_set_size()
                        + geom_text(aes(label=..count..), hjust=1.1, stat='count')
                        + expand_limits(y=1000)),
                      queries = list(upset_query("E4 Female Up-regulated", fill = "red"),
                                     upset_query("E4 Male Up-regulated", fill = "red"),
                                     upset_query("E4 Female Down-regulated", fill = "blue"),
                                     upset_query("E4 Male Down-regulated", fill = "blue")),
                      base_annotations = list('Intersection size' = (
                        intersection_size(bar_number_threshold=1, width=0.5)
                        + scale_y_continuous(expand=expansion(mult=c(0, 0.05)),limits = c(0,800)) # space on top
                        + theme(
                              # hide grid lines
                              panel.grid.major=element_blank(),
                              panel.grid.minor=element_blank(),
                              # show axis lines
                              axis.line=element_line(colour='black')))),
                      stripes = upset_stripes(
                        geom=geom_segment(size=12),  # make the stripes larger
                        colors=c('grey95', 'white')),
                      # to prevent connectors from getting the colorured
                      # use `fill` instead of `color`, together with `shape='circle filled'`
                      matrix = intersection_matrix(
                        geom=geom_point(
                          shape='circle filled',
                          size=3,
                          stroke=0.45)),
                      sort_sets=FALSE,
                      sort_intersections='descending'
                    )
  upset_gene <- upset_gene + ggtitle(paste0(i,", adj_p_val < 0.05"))
  i <- gsub(" ","_",i)
  pdf(paste0(out, "DEGs/upset/",tolower(i),"_upset.pdf"), height = 6, width = 8)
  print(upset_gene)
  dev.off()
}